The major hypothesis guiding the research supported by this grant over the past 10 years has been the proposal that lipid products of phosphoinositide 3-kinase (PI3K) play a role in cell growth and survival and that overproduction of these lipids can contribute to cell transformation. In the past year it has been demonstrated that PI3K itself is an oncogene in chickens and in mice and that PI3K contributes to several cell survival pathways. Many questions remain. The specific hypothesis to be tested in this proposal is that phosphoinositides generated by PI3K act as nucleation sites for the recruitment of specific signaling proteins at subcompartmentsof cell membranes to initiatesignaling cascades. The particular phosphoinositide generated and its cellular location will determine the ultimate cellular response. Four specific aims will address this hypothesis: 1) We will delete genes for specific subunits of PI3K in mouse ES cells and use these cells to generate mice with homozygous deletions. We will also use cultures of these cells to investigate the importance of PI3K in signal transduction. Multipleisoforms of catalytic and regulatorysubunits exist and this approach will allow us to determine their importance. 2) We will further characterize proteins that interact with the lipid products of PI3K and investigate the ability of these proteins to rescue defects due to loss of PI3K. 3) We will develop an assay for the location of PI3K lipid products in live cells. These studies will provide new information about how PI3K transforms cells and will suggest new targets for pharmaceutical interventionin thispathway.